搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Importance Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility. Objective To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury. Design,Setting,and Subjects This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient,transgenic mouse model. Induced pluripotent stem cell-derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24,2015,to May 2,2016,and data were analyzed from April 7,2015,to May 27,2016. Interventions Human iPSCs were used to derive motor neurons in vitro before transplant. Main Outcomes and Measures Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point). Results In 13 of the 32 mice undergoing the experiment,human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation,54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation,33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found. Conclusions and Relevance Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury,including facial paralysis. Level of Evidence NA. View Publication

Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells. View Publication

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs),whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac,fetal liver,and adult bone marrow. However,HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also,cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus,GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow. View Publication

BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2,CD3,CD7,CD14,CD16,CD24,CD36,CD38,CD56,CD66b,or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that,in combination with other markers,allows easy isolation of the stem cell fraction at unprecedented purities. View Publication

BACKGROUND Aside from its importance in reproduction,estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However,the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). RESULTS E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-$$)-dependent pathway. During hematopoietic differentiation of hPSCs,ER-$$ is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-$$ positive cells. Interestingly,continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-$$ selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors,and subsequent erythropoiesis,whereas ER-$$ selective agonist did not. Furthermore,ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods,does E2 signaling potentiate hematopoietic development,suggesting universal function of E2 on hematopoiesis. CONCLUSIONS Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions. View Publication

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here,we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However,this effect is at the expense of HSC function,because continued Hh activation during regeneration represses expression of specific cell cycle regulators,leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo. View Publication

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and myeloid skewing. We report that culture of bone marrow (BM) HSCs from aged mice with epidermal growth factor (EGF) suppressed myeloid skewing,increased multipotent colony formation,and increased HSC repopulation in primary and secondary transplantation assays. Mice transplanted with aged,EGF-treated HSCs displayed increased donor cell engraftment within BM HSCs and systemic administration of EGF to aged mice increased HSC self-renewal capacity in primary and secondary transplantation assays. Expression of a dominant negative EGFR in Scl/Tal1 + hematopoietic cells caused increased myeloid skewing and depletion of long term-HSCs in 15-month-old mice. EGF treatment decreased DNA damage in aged HSCs and shifted the transcriptome of aged HSCs from genes regulating cell death to genes involved in HSC self-renewal and DNA repair but had no effect on HSC senescence. These data suggest that EGFR signaling regulates the repopulating capacity of aged HSCs. Subject areas: Human physiology,cellular physiology,molecular medicine,stem cells research,functional aspects of cell biology View Publication

An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives,including lung,liver,and pancreas,are of interest for regenerative medicine,but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds,two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm,a higher efficiency than that achieved by Activin A or Nodal,commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers,can participate in normal development when injected into developing embryos,and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives. View Publication